Vascular Endothelial Cells Produce Soluble Factors That Mediate the Recovery of Human Hematopoietic Stem Cells after Radiation Injury

AbstractThe risk of terrorism with nuclear or radiologic weapons is considered to be high over the coming decade. Ionizing radiation can cause a spectrum of hematologic toxicities, from mild myelosuppression to myeloablation and death. However, the potential regenerative capacity of human hematopoietic stem cells (HSCs) after radiation injury has not been well characterized. In this study, we sought to characterize the effects of ionizing radiation on human HSCs and to determine whether signals from vascular endothelial cells could promote the repair of irradiated HSCs. Exposure of human bone marrow CD34+ cells to 400 cGy caused a precipitous decline in hematopoietic progenitor cell content and primitive cells capable of repopulating nonobese diabetic/severe combined immunodeficient mice (SCID-repopulating cells), which was not retrievable via treatment with cytokines. Conversely, culture of 400 cGy–irradiated bone marrow CD34+ cells with endothelial cells under noncontact conditions supported the differential recovery of both viable progenitor cells and primitive SCID-repopulating cells. These data illustrate that vascular endothelial cells produce soluble factors that promote the repair and functional recovery of HSCs after radiation injury and suggest that novel factors with radiotherapeutic potential can be identified within this milieu

Pathogenic Mutations Differentially Regulate Cell-to-Cell Transmission of α-Synuclein

Recent studies suggest that the cell-to-cell spread of pathological α-synuclein (α-syn) plays important roles in the development of Parkinson’s disease (PD). PD patients who carry α-syn gene mutations often have an earlier onset and more severe clinical symptoms and pathology than sporadic PD cases who carry the wild-type (WT) α-syn gene. However, the molecular mechanism by which α-syn gene mutations promote PD remains unclear. Here, we hypothesized that pathogenic mutations facilitate the intercellular transfer and cytotoxicity of α-syn, favoring an early disease onset and faster progression. We investigated the effects of eight known pathogenic mutations in human α-syn (A18T, A29S, A30P, E46K, H50Q, G51D, A53E, and A53T) on its pathological transmission in terms of secretion, aggregation, intracellular level, cytotoxicity, seeding, and induction of neuroinflammation in SH-SY5Y neuroblastoma cells, cultured rat neurons, and microglia, and the rat substantia nigra pars compacta. We found that 2 of the 8 mutations (H50Q and A53T) significantly increased α-syn secretion while 6 mutations (A18T, A29S, A30P, G51D, A53E, and E46K) tended to enhance it. In vitroα-syn aggregation experiments showed that H50Q promoted while G51D delayed aggregation most strongly. Interestingly, 3 mutations (E46K, H50Q, and G51D) greatly increased the intracellular α-syn level when cultured cells were treated with preformed α-syn fibrils (PFFs) compared with the WT, while the other 5 had no effect. We also demonstrated that H50Q, G51D, and A53T PFFs, but not E46K PFFs, efficiently seeded in vivo and acutely induced neuroinflammation in rat substantia nigra pars compacta. Our data indicate that pathogenic mutations augment the prion-like spread of α-syn at different steps and blockade of this pathogenic propagation may serve as a promising therapeutic intervention for PD

Frequency-dependent ERK phosphorylation in spinal neurons by electric stimulation of the sciatic nerve and the role in electrophysiological activity

The phosphorylation of extracellular signal-regulated kinase (pERK) in DRG and dorsal horn neurons is induced by the C-fiber electrical stimulation to the peripheral nerve. The present study was designed to investigate the expression and modulation of pERK in the rat dorsal horn neurons produced by repetitive electrical stimulation, and its involvement in the electrophysiological activity of dorsal horn neurons. Electrical stimulation of C-fiber intensity at different frequencies was applied to the sciatic nerve; the stimuli-induced pERK expression and the activity in dorsal horn neurons were studied by immunohistochemistry and extracellular recording, respectively. Electrical stimulation of C-fibers (3 mA) induced pERK expression in dorsal horn neurons in a frequency-dependent manner, indicating that the frequency of electrical stimulation is an important factor which activates the intracellular signal pathway in the spinal cord. To demonstrate the underlying mechanism of this frequency-dependent pERK expression, an NMDA receptor antagonist, MK-801, and a voltage sensitive calcium channel antagonist, nifedipine, were administrated intrathecally before the stimulation. We found that high frequency (0.5 Hz and 10 Hz) but not low frequent (0.05 Hz) stimulus-evoked pERK was partially inhibited by MK-801. Both high and low frequency stimulus-evoked pERK were inhibited by the nifedipine treatment. The extracellular single unit activities were recorded from the laminae I-II and V of the L4-5 dorsal horn, and we found that blockage of the intracellular ERK signal suppressed the wind-up responses in a dose-dependent manner. In contrast, any change in the mechanically evoked responses was not observed following the administration of ERK inhibitor. These observations indicate that ERK activation plays an important role in the induction of the wind-up responses in dorsal horn nociceptive neurons

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32\ua0mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by\ua0synthetic biology

Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4′- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology

Dynamic Tracking of Injected Mesenchymal Stem Cells after Myocardial Infarction in Rats: A Serial 7T MRI Study

Purpose. To track the fate of micron-sized particles of iron oxide (MPIO) labeled mesenchymal stem cells (MSCs) in vivo in a rat myocardial infarction model using 7T magnetic resonance imaging (MRI) scanner. Materials and Methods. Male MSCs (2 × 106/50 μL) dual-labeled with MPIO and CM-DiI were injected into the infarct periphery 7 days after myocardial infarction (MI). The control group received cell-free media injection. The temporal stem cell location, signal intensity, and cardiac function were dynamically assessed using a 7T MRI at 24 h before transplantation (baseline), 3 days, 2 weeks, and 4 weeks after transplantation, respectively. Results. MR hypointensities caused by MPIOs were observed on T2⁎-weighted images at all time points after MSCs injection. Cine-MRI showed that MSCs moderated progressive left ventricular remodeling. Double staining for iron and CD68 revealed that most of the iron-positive cells were CD68-positive macrophages. Real-time PCR for rat SRY gene showed the number of survival MSCs considerably decreased after transplantation. MSC-treated hearts had significantly increased capillary density in peri-infarct region and lower cardiomyocytes apoptosis and fibrosis formation. Conclusions. Iron particles are not a reliable marker for in vivo tracking the long-term fate of MSCs engraftment. Despite of poor cell retention, MSCs moderate left ventricular remodeling after MI

spd1672, a novel in vivo-induced gene, affects inflammatory response in a murine model of Streptococcus pneumoniae infection

spd1672, a novel Streptococcus pneumoniae (S. pn) gene induced in vivo, has been identified to contribute to the virulence of S. pn; however, the role of spd1672 during host innate immune reaction against S. pn infection is unknown. In the present study, mice were infected with wild type D39 and mutant D39Δspd1672 strains. Compared to the D39-infected mice, reduced bacterial load and attenuated inflammatory response were observed in the D39Δspd1672-treated mice. The levels of proinflammatory cytokines, including those of IFN-γ, TNF-α, and IL-1β, in the blood of D39Δspd1672-infected mice were lower than that in the D39-infected group. Additionally, attenuated activation of STAT3 and AKT was observed in the D39Δspd1672-infected mice. In conclusion, our data indicated that spd1672 expression modulates the release of proinflammatory cytokines, and AKT-STAT3 signaling appears to participate in the process. In conclusion, the present study extends our understanding of the role of an in vivo-induced gene in S. pn-host interaction.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author